ABSTRACTThe NLRP3 inflammasome is the most characterized inflammasome activated by cellular infection or stress, which is responsible for the maturation of proinflammatory cytokines IL-1β and IL-18. The precise molecular mechanism for negative regulation of NLRP3 inflammasome activation needs to be further defined. Here we identify leucine-rich repeat Fli-I-interacting protein 2 (LRRFIP2) as an NLRP3-associated protein and an inhibitor for NLRP3 inflammasome activation. LRRFIP2 binds to NLRP3 via its N terminus upon NLRP3 inflammasome activation, and also interacts with Flightless-I, a pseudosubstrate of caspase-1, via its Coil motif. Knockdown of Flightless-I significantly promotes NLRP3 inflammasome activation. LRRFIP2 enhances the interaction between Flightless-I and caspase-1, facilitating the inhibitory effect of Flightless-I on caspase-1 activation. Furthermore, silencing of Flightless-I abrogates the inhibitory effect of LRRFIP2 on NLRP3 inflammasome. These data demonstrate that LRRFIP2 inhibits NLRP3 inflammasome activation by recruiting the caspase-1 inhibitor Flightless-I, thus outlining a new mechanism for negative regulation of NLRP3 inflammasome.

f2: LRRFIP2 interacts with NLRP3 via its N-terminal motif.(a) Macrophages were primed with LPS and treated with ATP, Nigericin (Nig.) for the indicated times, immunoprecipitated with anti-LRRFIP2, and then immunobloted using the indicated antibodies. (b) 293T cells were co-transfected with expression plasmids for NLRP3-Myc, ASC-HA and LRRFIP2-Flag as indicated, immunoprecipitated with anti-Flag, and immunobloted using indicated antibodies. (c) LRRFIP2-truncated mutant vectors were constructed. (d) 293T cells were co-transfected with Myc-tagged NLRP3 and Flag-tagged LRRFIP2 truncated mutants, immunoprecipitated with anti-Flag and immunobloted using indicated antibodies. (e) 293T cells were co-transfected with Flag-tagged LRRFIP2 and Myc-tagged NLRP3 truncated mutants, immunoprecipitated with anti-Flag and immunobloted using indicated antibodies. Results are representative of three separate experiments with similar results. SN, supernatant.

Mentions:
Then, we identified the expression pattern of LRRFIP2 in macrophages (Supplementary Fig. S5, bottom). LPS robustly upregulated LRRFIP2 expression after 8 h. Although LRRFIP2 expression could be significantly induced by LPS, it did not bind to NLRP3 in macrophages treated with LPS only (Supplementary Fig. S5). Next, we investigated whether LRRFIP2 could indeed interact with NLRP3 after inflammasome activation. Immunoprecipitation assay showed that LRRFIP2 co-precipitated with NLRP3 and other components 10 min after ATP stimuli and 30 min after Nigericin stimuli (Fig. 2a). Therefore, LRRFIP2 could interact with the NLRP3 inflammasome complex very rapidly.

f2: LRRFIP2 interacts with NLRP3 via its N-terminal motif.(a) Macrophages were primed with LPS and treated with ATP, Nigericin (Nig.) for the indicated times, immunoprecipitated with anti-LRRFIP2, and then immunobloted using the indicated antibodies. (b) 293T cells were co-transfected with expression plasmids for NLRP3-Myc, ASC-HA and LRRFIP2-Flag as indicated, immunoprecipitated with anti-Flag, and immunobloted using indicated antibodies. (c) LRRFIP2-truncated mutant vectors were constructed. (d) 293T cells were co-transfected with Myc-tagged NLRP3 and Flag-tagged LRRFIP2 truncated mutants, immunoprecipitated with anti-Flag and immunobloted using indicated antibodies. (e) 293T cells were co-transfected with Flag-tagged LRRFIP2 and Myc-tagged NLRP3 truncated mutants, immunoprecipitated with anti-Flag and immunobloted using indicated antibodies. Results are representative of three separate experiments with similar results. SN, supernatant.

Mentions:
Then, we identified the expression pattern of LRRFIP2 in macrophages (Supplementary Fig. S5, bottom). LPS robustly upregulated LRRFIP2 expression after 8 h. Although LRRFIP2 expression could be significantly induced by LPS, it did not bind to NLRP3 in macrophages treated with LPS only (Supplementary Fig. S5). Next, we investigated whether LRRFIP2 could indeed interact with NLRP3 after inflammasome activation. Immunoprecipitation assay showed that LRRFIP2 co-precipitated with NLRP3 and other components 10 min after ATP stimuli and 30 min after Nigericin stimuli (Fig. 2a). Therefore, LRRFIP2 could interact with the NLRP3 inflammasome complex very rapidly.

ABSTRACTThe NLRP3 inflammasome is the most characterized inflammasome activated by cellular infection or stress, which is responsible for the maturation of proinflammatory cytokines IL-1β and IL-18. The precise molecular mechanism for negative regulation of NLRP3 inflammasome activation needs to be further defined. Here we identify leucine-rich repeat Fli-I-interacting protein 2 (LRRFIP2) as an NLRP3-associated protein and an inhibitor for NLRP3 inflammasome activation. LRRFIP2 binds to NLRP3 via its N terminus upon NLRP3 inflammasome activation, and also interacts with Flightless-I, a pseudosubstrate of caspase-1, via its Coil motif. Knockdown of Flightless-I significantly promotes NLRP3 inflammasome activation. LRRFIP2 enhances the interaction between Flightless-I and caspase-1, facilitating the inhibitory effect of Flightless-I on caspase-1 activation. Furthermore, silencing of Flightless-I abrogates the inhibitory effect of LRRFIP2 on NLRP3 inflammasome. These data demonstrate that LRRFIP2 inhibits NLRP3 inflammasome activation by recruiting the caspase-1 inhibitor Flightless-I, thus outlining a new mechanism for negative regulation of NLRP3 inflammasome.